The human intestinal microbiota contains more than 35 species of the order Bacteroidales, which collectively are the most abundant Gram negative bacteria of this microbial ecosystem. We know very little about how these intestinal bacteria interact with each other to establish health-promoting microbial communities. This application addresses antagonistic interactions mediated by Type VI secretion systems (T6SS) of the gut Bacteroidales. Until recently, T6SS were only described in Proteobacterial species. We have found that T6SS are abundant in the gut Bacteroidales and we have identified 114 T6SS loci in 205 human gut Bacteroidales genomes. We have experimentally demonstrated that two of these T6SSs mediate antagonistic interactions with other Bacteroidales species. The two aims of this proposal are designed to study the most important questions regarding these T6SSs in terms of ecosystem establishment, defense, invasion and stability. In Aim 1, we will take a molecular approach to study the T6SSs, their range of bacterial targeting, identification of toxic effectors, the basis of theT6SS resistance observed in some strains, and regulation of T6SS firing by a transcriptional repressor of the TetR family. We will also analyze if T6SS of gut Bacteroidales target enteric pathogens, thereby contributing to the microbiota's colonization resistance function. The second aim is designed to utilize the mutants and molecular tools that were generated in the first aim to comprehensively study the relevance of T6SS-mediated antagonism in the mammalian gut. For this aim, we will perform experiments using a gnotobiotic mouse intestinal colonization model to determine if T6SSs facilitate the invasion of a strain into an established ecosystem, and the roles of T6SSs in thwarting colonization of competing sensitive strains. These studies will also include analyses of T6SS-mediated antagonism between naturally co- resident strains from a human gut ecosystems to determine if T6SS-mediated antagonism occurs in a naturally stable human gut microbial community. In addition, we will study the ability of T6SS to be transferred by integrative conjugative elements between strains in vitro and within the gnotobiotic mouse colon. These studies will contribute significantl to our understanding the dynamic interactions that occur between gut species and the factors that contribute to the establishment of human intestinal microbial ecosystems.